EP3131575B1 - Vaccin contre un streptocoque du groupe a - Google Patents

Vaccin contre un streptocoque du groupe a Download PDF

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EP3131575B1
EP3131575B1 EP15779482.7A EP15779482A EP3131575B1 EP 3131575 B1 EP3131575 B1 EP 3131575B1 EP 15779482 A EP15779482 A EP 15779482A EP 3131575 B1 EP3131575 B1 EP 3131575B1
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Prior art keywords
seq
peptide
variant
spycep
fragment
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EP3131575A1 (fr
EP3131575A4 (fr
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Manisha Pandey
Michael BATZLOFF
Michael Good
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Griffith University
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Griffith University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092—Streptococcus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4873—Cysteine endopeptidases (3.4.22), e.g. stem bromelain, papain, ficin, cathepsin H
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1275—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Streptococcus (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/22—Cysteine endopeptidases (3.4.22)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • Embodiments not according to the invention relate to the prevention and treatment of infectious diseases. More particularly, embodiments not according to the invention relate to a vaccine for treating or preventing group A streptococcus streptococcus-associated diseases and conditions.
  • the invention is set out in the appended set of claims.
  • Vaccines against Streptococcus pyogenes have long been sought due to the debilitating diseases caused by the bacterium, particularly rheumatic fever and rheumatic heart disease.
  • Rheumatic fever is an uncommon disease today in most developed countries but it remains the major cause of acquired heart disease in children, adolescents and young adults in the developing world.
  • invasive GAS disease is a frequent cause of sepsis in children and adults and has a high-case fatality rate.
  • post-streptococcal glomerulonephritis which likely contributes to the high rates of end-stage renal failure in many GAS endemic regions.
  • GAS pharyngitis and impetigo are responsible for the greatest absolute number of GAS infections each year. GAS pharyngitis affects approximately 8%-15% of school-aged children per year and GAS impetigo is a very common infection in children prevalence of 10-50%. Not only are severe GAS-associated diseases a problem in developing countries, but even in developed countries particularly virulent GAS strains have emerged that are resistant to standard antibiotic therapies and cause debilitating diseases such as severe necrotizing fasciitis.
  • M protein An important virulence factor of GAS is M protein, which is strongly anti-phagocytic and binds to serum factor H, destroying C3-convertase and preventing opsonization by C3b.
  • Vaccines have been developed that contain immunogenic peptides from the conserved C-repeat portion of the M protein, such as T and B-cell epitopes from the C-repeat region or the J8 and J14 peptide vaccines that contain single, minimal B cell epitopes from this C-repeat region.
  • Hayman et al. International Immunology, vol. 9, no. 11, pp. 1723-1733 , deals with mapping the minimal murine T cell and B cell epitopes within a peptide vaccine candidate from the conserved region of the M protein of group A streptococcus.
  • WO 2010/076618 A1 describes compositions useful for reducing the risk of preventing, and/or treating S. pyogenes (GAS) infections which comprise combinations of GAS antigens, nucleic acid molecules encoding the antigens, or antibodies which specifically bind to the antigens.
  • GAS S. pyogenes
  • Pandey et al. The Journal of Immunology, 2015, 194:5915-5925 , deals with a synthetic M protein peptide that synergizes with a CXC Chemokine Protease to induce Vaccine-mediated protection against virulent Streptococcal Pyoderma and Bacteremia.
  • WO 2009/034472 A2 describes mutants of GAS57 which are unable to cleave IL-8 and similar substrates but which still maintain the ability to induce protection against S. pyogenes and describes antibodies which specifically bind to GAS57 and which inhibit its ability to cleave IL-8 and similar substrates.
  • opsonisation assays use whole blood as a source of neutrophils and complement, it was not known that neutrophils were required for in vivo protection. Furthermore, opsonisation assays demonstrate relatively modest reductions in CFU (typically less than 10-fold), whereas J8-induced protection in vivo can result in several log order reductions in bacterial bio-burden. More particularly, the inventors now show that neutrophil inhibitors such as SpyCEP which inactivates the neutrophil chemotactic agent interleukin 8 work against J8 in inducing immunity to group A streptococcus.
  • neutrophil inhibitors such as SpyCEP which inactivates the neutrophil chemotactic agent interleukin 8 work against J8 in inducing immunity to group A streptococcus.
  • An embodiment not according to the invention relates to restoring or enhancing neutrophil activity to thereby assist M protein-induced immunity to group A streptococcus.
  • An embodiment not according to the invention provides a method of eliciting an immune response to group A streptococcal bacteria in a mammal, said method including the step of administering to the mammal: an M protein fragment, variant or derivative thereof; and an agent that facilitates restoring or enhancing neutrophil activity; to thereby elicit an immune response to group A streptococcal bacteria in the mammal.
  • An embodiment not according to the invention provides a method of immunizing a mammal against group A streptococcal bacteria, said method including the step of administering to the mammal: an M protein, fragment, variant or derivative thereof; and an agent that facilitates restoring or enhancing neutrophil activity, to thereby immunize the mammal against group A streptococcal bacteria.
  • An embodiment not according to the invention provides a method of treating or preventing a group A streptococcal bacterial infection in a mammal, said method including the step of administering to the mammal an M protein fragment, variant or derivative thereof, or an antibody or antibody fragment thereto; and an agent that facilitates restoring or enhancing neutrophil activity; to thereby treat or prevent the group A streptococcal bacterial infection in the mammal.
  • a further aspect provides a composition suitable for administration to a mammal, said composition comprising: an M protein fragment, variant or derivative thereof, or an antibody or antibody fragment thereto; and an agent that facilitates restoring or enhancing neutrophil activity as defined in the appended claims.
  • Embodiments not according to the invention relate to administration of one or more isolated nucleic acids encoding an M protein fragment, variant or derivative thereof and an agent that facilitates restoring or enhancing neutrophil activity or a composition comprising same.
  • the invention is set out in the appended set of claims.
  • the M protein fragment is or comprises a conserved region of the M protein.
  • the fragment is an immunogenic fragment that, in one embodiment, consisting of the p145 peptide of SEQ ID NO: 56.
  • the immunogenic fragment comprises the J8 peptide of SEQ ID NO: 58.
  • the immunogenic M protein fragment comprises the J8 peptide of SREAKKQSREAKKQVEKALKQVEKALC (SEQ ID NO: 59); SREAKKQSREAKKQVEKALKQSREAKC (SEQ ID NO: 60); SREAKKQVEKALKQSREAKKQVEKALC (SEQ ID NO: 61); or SREAKKQVEKALDASREAKKQVEKALC (SEQ ID NO:62).
  • the agent that facilitates restoring or enhancing neutrophil activity is a protein, or a fragment thereof, that normally directly or indirectly inhibits or suppresses neutrophils or neutrophil activity.
  • administration of the protein or a fragment thereof elicits an immune response to the protein and/or to group A streptococcus.
  • the agent that facilitates restoring or enhancing neutrophil activity is an antibody or antibody fragment which binds a protein, or a fragment thereof, that normally directly or indirectly inhibits or suppresses neutrophils or neutrophil activity.
  • the agent that facilitates restoring or enhancing neutrophil activity comprises the SpyCEP peptide of NSDNIKENQFEDFDEDWENF (SEQ ID NO: 18) or a SpyCEP peptide variant consisting of an amino acid sequence at least 90% identical to SEQ ID NO: 18.
  • an isolated peptide comprises, consists essentially of, or consists of the amino acid sequence selected from the group consisting of NSDNIKENQFEDFDEDWENF (SEQ ID NO: 18); SREAKKQSREAKKQVEKALKQVEKALC (SEQ ID NO: 59); SREAKKQSREAKKQVEKALKQSREAKC (SEQ ID NO: 60); SREAKKQVEKALKQSREAKKQVEKALC (SEQ ID NO: 61); and SREAKKQVEKALDASREAKKQVEKALC (SEQ ID NO: 62); or a fragment or variant thereof.
  • the following embodiments are not according to the invention and describe an isolated nucleic acid encoding the isolated peptide of the aforementioned aspect, a genetic construct comprising the isolated nucleic acid and/or a host cell comprising the genetic construct.
  • the following embodiment is not according to the invention and describes an antibody or antibody fragment which binds or is raised against the isolated peptide of the aforementioned aspect.
  • composition comprising the isolated peptide, the isolated nucleic acid, the genetic construct, the host cell and/or the antibody or antibody fragment of the previous aspects.
  • neutrophil activity is important for successful immunization with J8 peptide against group A streptococci. More particularly, it has been realized that certain proteases of group A streptococci such as SpyCEP exert a deleterious or suppressive effect on neutrophils by proteolytically inactivating the neutrophil chemotactic agent interleukin 8. It is therefore described but not according to the invention that by immunizing with J8 peptide, or other M protein fragment, and also SpyCEP, an immune response will be elicited to SpyCEP which at least partly reduces the ability of SpyCEP to inactivate interleukin 8 and thereby suppress the neutrophil response.
  • group A streptococci such as SpyCEP exert a deleterious or suppressive effect on neutrophils by proteolytically inactivating the neutrophil chemotactic agent interleukin 8. It is therefore described but not according to the invention that by immunizing with J8 peptide, or other M protein fragment, and also SpyCEP
  • anti-SpyCEP antibodies may be therapeutically administered to thereby elicit an enhanced immune response to J8 peptide.
  • an immunodominant epitope of SpyCEP has been identified.
  • the invention is, in one embodiment, for use in a method of treating or preventing a group A streptococcal bacterial infection in a mammal, by particularly virulent strains or isolates of Group A streptococci that are resistant to the typical antibiotic treatments used for group A streptococcal infections. These strains or isolates typically cause serious infections of the skin ( e.g . necrotizing fasciitis) and in some cases may harbour a CovR/SCovR/S mutation.
  • Described herein, but not according to the invention, is to administer an M protein fragment, variant or derivative thereof and an agent that facilitates restoring or enhancing neutrophil activity to a mammal to thereby elicit an immune response in the mammal.
  • isolated material that has been removed from its natural state or otherwise been subjected to human manipulation.
  • Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state.
  • Isolated material may be in native, chemical synthetic or recombinant form.
  • protein is meant an amino acid polymer.
  • the amino acids may be natural or non-natural amino acids, D- or L-amino acids as are well understood in the art.
  • protein includes and encompasses " peptide " , which is typically used to describe a protein having no more than fifty (50) amino acids and " polypeptide " , which is typically used to describe a protein having more than fifty (50) amino acids.
  • a “frag" is a segment, domain, portion or region of a protein (such as M protein, p145, J14 or J8 or SpyCEP or a SpyCEP peptide or epitope), which constitutes less than 100% of the amino acid sequence of the protein. It will be appreciated that the fragment may be a single fragment or may be repeated alone or with other fragments.
  • a protein such as M protein, p145, J14 or J8 or SpyCEP or a SpyCEP peptide or epitope
  • fragments may comprise, consist essentially of or consist of up to 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 100, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550 or 1600 amino acids of the full length protein.
  • the fragment according to the present invention is an immunogenic fragment.
  • immunogenic indicates the ability or potential to generate or elicit an immune response, such as to Group A strep or molecular components thereof such as M protein, upon administration of the immunogenic fragment to a mammal. Described herein, but not according to the invention, the immune response elicited by the immunogenic fragment is protective.
  • elicit an immune response is meant generate or stimulate the production or activity of one or more elements of the immune system inclusive of the cellular immune system, antibodies and/or the native immune system.
  • the one or more elements of the immune system include B lymphocytes, antibodies and neutrophils.
  • the terms “ immunize” , “ vaccinate” and “ vaccine” refer to methods and/or compositions that elicit a protective immune response against Group A Strep, whereby subsequent infection by Group A Strep is at least partly prevented or minimized.
  • group A streptococcus As used herein the terms " group A streptococcus " , “ Group A Streptococci” , “ Group A Streptococcal “ , “ Group A Strep” and the abbreviation “ GAS” refer to streptococcal bacteria of Lancefield serogroup A which are gram positive ⁇ -hemolytic bacteria of the species Streptococcus pyogenes.
  • An important virulence factor of GAS is M protein, which is strongly anti-phagocytic and binds to serum factor H, destroying C3-convertase and preventing opsonization by C3b.
  • These also include virulent "mutants” such as CovR/S or CovRS mutants such as described in Graham et al., 2002, PNAS USA 99 13855 .
  • Group A streptococci Diseases and conditions caused by group A streptococci include cellulitis, erysipelas, impetigo, scarlet fever, throat infections such as acute pharyngitis ("strep throat"), bacteremia, toxic shock syndrome, necrotizing fasciitis, acute rheumatic fever and acute glomerulonephritis.
  • strep throat acute pharyngitis
  • bacteremia bacteremia
  • toxic shock syndrome necrotizing fasciitis
  • acute rheumatic fever acute glomerulonephritis.
  • neutrophils or neutrophil granulocytes are cells that form part of the polymorphonuclear cell family (PMNs) together with basophils and eosinophils.
  • Neutrophils are relatively short-lived phagocytic cells formed from bone marrow stem cells and typically constitute 40% to 75% of white blood cells in mammals.
  • phagocytic neutrophils release soluble antimicrobials (e.g. granule proteins) and generate neutrophil extracellular traps.
  • Neutrophils are responsive to molecules such as interleukin-8 (IL-8), C5a, fMLP and leukotriene B4 which promote neutrophil chemotaxis to sites of injury and/or acute inflammation.
  • IL-8 interleukin-8
  • C5a C5a
  • fMLP fMLP
  • leukotriene B4 leukotriene B4 which promote neutrophil chemotaxis to sites of injury and/or acute inflammation.
  • an " agent that facilitates restoring or enhancing neutrophil activity" is a molecule that directly or indirectly at least partly increases, enhances or restores the production, migration and/or chemotaxis of neutrophils and/or one or more immunological activities of neutrophils.
  • it is for use in a method of eliciting an immune response to group A streptococcal bacteria in a mammal. It is described herein, but not according to the invention that the agent binds and at least partly inactivates the neutrophil inhibitor.
  • the neutrophil inhibitor may be a molecule derived or originating from Group A Streptococcal bacteria.
  • the neutrophil inhibitor is a serine protease, or a fragment thereof, that proteolytically cleaves interleukin 8. It is described herein, but not according to the invention, that the neutrophil inhibitor is SpyCEP or a fragment thereof.
  • SpyCEP is a 170-kDa multidomain serine protease expressed on the surface of the human pathogen Streptococcus pyogenes, which plays an important role in infection by catalyzing cleavage and inactivation of the neutrophil chemoattractant interleukin-8. Examples of SpyCEP amino acid sequences may be found under accession numbers YP597949.1 and ( S .
  • the agent that facilitates restoring or enhancing neutrophil activity is the SpyCEP peptide of SEQ ID NO: 18.
  • the agent that facilitates restoring or enhancing neutrophil activity is an antibody or antibody fragment that binds said SpyCEP peptide. with the amino acid sequence set forth in SEQ ID NO: 18 (NSDNIKENQFEDFDEDWENF).
  • an anti-SEQ ID NO: 18 peptide antiserum can block the degradation of IL8 as effectively as anti-rSpyCEP antibodies.
  • SEQ ID NO: 18 is, or comprises, the dominant epitope on SpyCEP that can induce functional antibodies.
  • an " M protein fragment” is any fragment as defined in the appended set of claims. As described herein but not according to the invention, the " M protein fragment " is any fragment of a GAS M protein that is immunogenic and/or is capable of being bound by an antibody or antibody fragment. Typically, the fragment is, comprises, or is contained within an amino acid sequence of a C-repeat region of a GAS M protein, or a fragment thereof. In one embodiment of the compositiosn of the present invention, the " M protein fragment " consists of p145 which is a 20mer with the amino acid sequence LRRDLDASREAKKQVEKALE (SEQ ID NO: 56). In this regard, but not according to the invention, fragments of the p145 amino acid sequence may be present in J14 or J8 peptides.
  • J14 peptide may comprise the amino acid sequence KQAEDKVK ASREAKKQVEKALE QLEDRVK (SEQ ID NO: 57) or a fragment or variant thereof, a peptide with minimal B and T cell epitopes within p145 was identified as a GAS M protein C-region peptide devoid of potentially deleterious T cell autoepitopes, but which contained an opsonic B cell epitope.
  • J14 is a chimeric peptide that contains 14 amino acids from M protein C-region (shown in bold ) and is flanked by yeast-derived GCN4 sequences which was necessary to maintain the correct helical folding and conformational structure of the peptide.
  • J8 peptide is a peptide which comprises an amino acid sequence at least partly derived from, or corresponding to, a GAS M protein C-region peptide.
  • J8 peptide suitably comprises a conformational B-cell epitope and lacks potentially deleterious T-cell autoepitopes.
  • the immunogenic M protein fragment is a J8 peptide amino acid sequence of QAEDKVKQ SREAKKQVEKAL KQLEDKVQ (SEQ ID NO: 58), wherein the bolded residues correspond to residues 344 to 355 of the GAS M protein.
  • J8 is a chimeric peptide that further comprises flanking GCN4 DNA-binding protein sequences which assist maintaining the correct helical folding and conformational structure of the J8 peptide.
  • a protein " variant" shares a definable nucleotide or amino acid sequence relationship with a reference amino acid sequence.
  • the reference amino acid sequence may be an amino acid sequence of M protein, SpyCEP or a fragment of these, as hereinbefore described.
  • the " variant " protein may have one or a plurality of amino acids of the reference amino acid sequence deleted or substituted by different amino acids.
  • protein variants share at least 70% or 75%, at least 80% or 85%, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with a reference amino acid sequence.
  • the SpyCEP peptide variant consisting of an amino acid sequence at least 90% identical to SEQ ID NO: 18or the SpyCEP peptide of SEQ ID NO: 18further consists of one or a plurality of lysine residues at an N- and/or C-terminus thereof.
  • the plurality of lysine residues e.g . polylysine
  • compositions of the present invention are:
  • a model peptide can be synthesised to fold to this conformation.
  • the inventors designed a model ⁇ -helical coiled coil peptide based on the structure of the GCN4 leucine zipper (O'Shea et al., 1991).
  • the first heptad contains the sequence MKQLEDK (SEQ ID NO: 63), which includes several of the features found in a stable coiled coil heptad repeat motif ( a-b-c-d-e-f-g ) n (Cohen & Parry, 1990).
  • the GCN4 peptide also contains a consensus valine in the a position. It has also been noted that when positions a and d are occupied by V and L a coiled coil dimer is favoured (Harbury et al., 1994).
  • a model heptad repeat was derived from these consensus features of the GCN4 leucine zipper peptide: (VKQLEDK; SEQ ID NO: 64) with the potential to form a ⁇ -helical coiled coil.
  • This peptide became the framework peptide.
  • Overlapping fragments of a conformational epitope under study were embedded within the model coiled coil peptide to give a chimeric peptide.
  • Amino acid substitutions, designed to ensure correct helical coiled coil conformations (Cohen & Parry, 1990) were incorporated into the chimeric peptides whenever an identical residue was found in both the helical model peptide and the epitope sequence.
  • sequence comparisons are typically performed by comparing sequences over a "comparison window” to identify and compare local regions of sequence similarity.
  • a “ comparison window” refers to a conceptual segment of typically 6, 9 or 12 contiguous residues that is compared to a reference sequence.
  • the comparison window may comprise additions or deletions (i.e ., gaps) of about 20% or less as compared to the reference sequence for optimal alignment of the respective sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (Geneworks program by Intelligenetics; GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA,) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.
  • sequence identity is used herein for illustration purposes only in its broadest sense to include the number of exact nucleotide or amino acid matches having regard to an appropriate alignment using a standard algorithm, having regard to the extent that sequences are identical over a window of comparison.
  • a “ percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g ., A, T, C, G, I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison ( i.e ., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • sequence identity may be understood to mean the "match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA).
  • derivatives are molecules such as proteins, fragments or variants thereof that have been altered, for example by conjugation or complexing with other chemical moieties, by post-translational modification (e.g . phosphorylation, or acetylation), modification of glycosylation (e.g . adding, removing or altering glycosylation), lipidation and/or inclusion of additional amino acid sequences as would be understood in the art.
  • an additional amino acid sequence may comprise one or a plurality of lysine residues at an N and/or C-terminus thereof.
  • the plurality of lysine residues e.g . polylysine
  • lipid core peptide One particular J8 peptide derivative described in Olive et al., 2002, Infect & Immun. 70 2734 is a "lipid core peptide". It is described herein, but not according to the invention, that a lipid core peptide may comprise a plurality of J8 peptides ( e.g. four J8 peptides) synthesized directly onto two amino groups of each lysine of a branched polylysine core coupled to a lipophilic anchor.
  • Additional amino acid sequences may include fusion partner amino acid sequences which create a fusion protein.
  • fusion partner amino acid sequences may assist in detection and/or purification of the isolated fusion protein.
  • metal-binding e.g . polyhistidine
  • MBP maltose binding protein
  • GST glutathione S-transferase
  • GFP fluorescent protein sequences
  • epitope tags such as myc, FLAG and haemagglutinin tags.
  • immunogenic proteins, fragments and/or derivatives described herein may be produced by any means known in the art, including chemical synthesis, recombinant DNA technology and proteolytic cleavage to produce peptide fragments.
  • Chemical synthesis is inclusive of solid phase and solution phase synthesis. Such methods are well known in the art, although reference is made to examples of chemical synthesis techniques as provided in Chapter 9 of SYNTHETIC VACCINES Ed. Nicholson (Blackwell Scientific Publications ) and Chapter 15 of CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al., (John Wiley & Sons, Inc. NY USA 1995-2008 ) (not according to the invention). In this regard, reference is also made to International Publication WO 99/02550 and International Publication WO 97/45444 (not according to the invention).
  • Recombinant proteins may be conveniently prepared by a person skilled in the art using standard protocols as for example described in Sambrook et al., MOLECULAR CLONING. A Laboratory Manual (Cold Spring Harbor Press, 1989), in particular Sections 16 and 17 ; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al., (John Wiley & Sons, Inc. NY USA 1995-2008), in particular Chapters 10 and 16 ; and CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al., (John Wiley & Sons, Inc. NY USA 1995-2008), in particular Chapters 1, 5 and 6 (not according to the invention).
  • recombinant protein preparation includes expression of a nucleic acid encoding the protein in a suitable host cell.
  • nucleic acids encoding an M protein fragment, variant or derivative thereof and an agent that facilitates restoring or enhancing neutrophil activity or a composition comprising same to thereby elicit an immune response to GAS and/or immunize against a GAS infection.
  • nucleic acid designates single- or doublestranded DNA and RNA.
  • DNA includes genomic DNA and cDNA.
  • RNA includes mRNA, RNA, RNAi, siRNA, cRNA and autocatalytic RNA.
  • Nucleic acids may also be DNA-RNA hybrids.
  • a nucleic acid comprises a nucleotide sequence which typically includes nucleotides that comprise an A, G, C, T or U base. However, nucleotide sequences may include other bases such as modified purines (for example inosine, methylinosine and methyladenosine) and modified pyrimidines (for example, thiouridine and methylcytosine).
  • the one or more isolated nucleic acids encoding an M protein fragment, variant or derivative thereof and an agent that facilitates restoring or enhancing neutrophil activity are in the form of a genetic construct suitable for administration to a mammal such as a human. Further described herein, but not according to the invention, is that the genetic construct is suitable for DNA vaccination of a mammal such as a human.
  • the genetic construct is in the form of, or comprises genetic components of, a plasmid, bacteriophage, a cosmid, a yeast or bacterial artificial chromosome as are well understood in the art. Genetic constructs may also be suitable for maintenance and propagation of the isolated nucleic acid in bacteria or other host cells, for manipulation by recombinant DNA technology.
  • the genetic construct is an expression construct.
  • the expression construct comprises the one or more nucleic acids operably linked to one or more additional sequences in an expression vector.
  • An " expression vector" may be either a self-replicating extra-chromosomal vector such as a plasmid, or a vector that integrates into a host genome.
  • operably linked is meant that said additional nucleotide sequence(s) is/are positioned relative to the nucleic acid to initiate, regulate or otherwise control transcription.
  • Regulatory nucleotide sequences (not according to the invention) will generally be appropriate for the host cell or tissue where expression is required. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells.
  • said one or more regulatory nucleotide sequences may include, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters as known in the art are described herein, but not according to the invention.
  • the expression construct described herein, but not according to the invention may also include an additional nucleotide sequence encoding a fusion partner (typically provided by the expression vector) so that the recombinant protein is expressed as a fusion protein, as hereinbefore described.
  • the isolated nucleic acids encoding an M protein, fragment, variant or derivative thereof and the agent that facilitates restoring or enhancing neutrophil activity may be administered by way of separate expression constructs or may be present in the same expression construct (e.g. a multi-cistronic expression construct).
  • DNA vaccination is by way of one or more plasmid DNA expression constructs.
  • Plasmids typically comprise a viral promoter (such as SV40, RSV or CMV promoters). Intron A may be included to improve mRNA stability and thereby increase protein expression.
  • Plasmids may further include a multiple cloning site, a strong polyadenylation/transcription termination signal, such as bovine growth hormone or rabbit beta-globulin polyadenylation sequences.
  • the plasmid may further comprise Mason-Pfizer monkey virus cis-acting transcriptional elements (MPV-CTE) with or without HIV rev increased envelope expression.
  • MPV-CTE Mason-Pfizer monkey virus cis-acting transcriptional elements
  • Additional modifications that may improve expression include the insertion of enhancer sequences, synthetic introns, adenovirus tripartite leader (TPL) sequences and/or modifications to polyadenylation and/or transcription termination sequences.
  • TPL adenovirus tripartite leader
  • An example of a DNA vaccine plasmid is pVAC which is commercially available from Invivogen.
  • DNA vaccinology is DNA Vaccines, Methods and Protocols, Second Edition (Volume 127 of Methods in Molecular Medicine series, Humana Press, 2006 ) (not according to the invention).
  • treating " , " treat “ or “ treatment” refers to a therapeutic intervention that at least partly ameliorates, eliminates or reduces a symptom or pathological sign of a Group A strep-associated disease, disorder or condition after it has begun to develop. Treatment need not be absolute to be beneficial to the subject. The beneficial effect can be determined using any methods or standards known to the ordinarily skilled artisan.
  • preventing " , " prevent “ or “ prevention” refers to a course of action initiated prior to infection by, or exposure to, group A strep and/or before the onset of a symptom or pathological sign of a Group A strep-associated disease, disorder or condition, so as to prevent infection and/or reduce the symptom or pathological sign. It is to be understood that such preventing need not be absolute to be beneficial to a subject.
  • a “ prophylactic " treatment is a treatment administered to a subject who does not exhibit signs of a group A strep-associated disease, disorder or condition, or exhibits only early signs for the purpose of decreasing the risk of developing a symptom or pathological sign of a Group A strep-associated disease, disorder or condition.
  • group A-strep-associated disease, disorder or condition any clinical pathology resulting from infection by group A strep and includes cellulitis, erysipelas, impetigo, scarlet fever, throat infections such as acute pharyngitis ("strep throat”), bacteremia, toxic shock syndrome, necrotizing fasciitis, acute rheumatic fever and acute glomerulonephritis.
  • group A-strep-associated disease, disorder or condition is meant any clinical pathology resulting from infection by group A strep and includes cellulitis, erysipelas, impetigo, scarlet fever, throat infections such as acute pharyngitis ("strep throat”), bacteremia, toxic shock syndrome, necrotizing fasciitis, acute rheumatic fever and acute glomerulonephritis.
  • the treatment and/or immunization methods disclosed herein include administration of the M protein fragment, variant or derivative and the agent that facilitates restoring or enhancing neutrophil activity to a mammal separately, or in combination, to thereby elicit an immune response by the mammal.
  • the treatment and/or immunization methods include administration of one or more isolated nucleic acids encoding an M protein fragment, variant or derivative thereof and an agent that facilitates restoring or enhancing neutrophil activity separately, or in combination, to thereby elicit an immune response by the mammal.
  • antibodies or antibody fragments to therapeutically treat GAS infections, such as by targeting SpyCEP at the site of infection (e.g. the skin). This may be performed in combination with M protein fragment immunization and/or administration of anti-M protein fragment antibodies or antibody fragments.
  • Antibodies and antibody fragments may be polyclonal or monoclonal, native or recombinant.
  • Antibody fragments include Fc, Fab or F(ab)2 fragments and/or may comprise single chain Fv antibodies (scFvs).
  • scFvs may be prepared, for example, in accordance with the methods described respectively in United States Patent No 5,091,513 , European Patent No 239,400 or the article by Winter & Milstein, 1991, Nature 349:293 .
  • Antibodies may also include multivalent recombinant antibody fragments, such as diabodies, triabodies and/or tetrabodies, comprising a plurality of scFvs, as well as dimerisation-activated demibodies ( e.g. WO/2007/062466 ).
  • multivalent recombinant antibody fragments such as diabodies, triabodies and/or tetrabodies, comprising a plurality of scFvs, as well as dimerisation-activated demibodies (e.g. WO/2007/062466 ).
  • such antibodies may be prepared in accordance with the methods described in Holliger et al., 1993 Proc Natl Acad Sci USA 90 6444 ; or in Kipriyanov, 2009 Methods Mol Biol 562 177 .
  • Anti-SpyCEP polyclonal antibodies may be obtained or purified from human sera from individuals exposed to, or infected by, Group A strep. Alternatively, polyclonal antibodies may be raised against purified or recombinant SpyCEP, or an immunogenic fragment thereof, in production species such as horses and then subsequently purified prior to administration.
  • Monoclonal antibodies may be produced using the standard method as for example, originally described in an article by Köhler & Milstein, 1975, Nature 256, 495 , or by more recent modifications thereof as for example, described in Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY, supra by immortalizing spleen or other antibody producing cells derived from a production species which has been inoculated with one or more of the isolated proteins, fragments, variants or derivatives described herein. Accordingly, monoclonal antibodies may be raised against an M protein fragment, variant or derivative and/or the agent that facilitates restoring or enhancing neutrophil activity (e.g. SpyCEP) for use as described herein.
  • SpyCEP the agent that facilitates restoring or enhancing neutrophil activity
  • the monoclonal antibody or fragment thereof may be in recombinant form. This may be particularly advantageous for "humanizing" the monoclonal antibody or fragment if the monoclonal antibody is initially produced by spleen cells of a non-human mammal.
  • a further aspect described herein, but not according to the invention, relates to therapeutic antibodies, wherein a M protein fragment may be a p145 peptide.
  • a fragment of SpyCEP may comprise or consist of the amino acid sequence NSDNIKENQFEDFDEDWENF (SEQ ID NO: 18).
  • the M protein fragment, variant or derivative and the agent that facilitates restoring or enhancing neutrophil activity, inclusive of antibodies or antibody fragments as disclosed herein may be administered to a mammal separately, or in combination, in the form of a composition.
  • composition comprises an acceptable carrier, diluent or excipient.
  • acceptable carrier diluent or excipient
  • diluent or excipient a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic administration.
  • a variety of carriers, diluent and excipients well known in the art may be used.
  • These may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates, water and pyrogen-free water.
  • sugars starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates, water and pyrogen-free
  • certain immunological agents may be used in combination with the J8 peptide, fragment, variant or derivative and the agent that facilitates restoring or enhancing neutrophil activity, or with one or more genetic constructs encoding these.
  • immunological agent includes within its scope carriers, delivery agents, immunostimulants and/or adjuvants as are well known in the art.
  • immunostimulants and adjuvants refer to or include one or more substances that enhance the immunogenicity and/or efficacy of a composition.
  • immunostimulants and adjuvants include squalane and squalene (or other oils of plant or animal origin); block copolymers; detergents such as Tween ® -80; Quil ® A, mineral oils such as Drakeol or Marcol, vegetable oils such as peanut oil; Corynebacterium-derived adjuvants such as Corynebacterium parvum; Propionibacterium-derived adjuvants such as Propionibacterium acne; Mycobacterium bovis (Bacille Calmette and Guerin or BCG); Bordetella pertussis antigens; tetanus toxoid; diphtheria toxoid; surface active substances such as hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethyldioctadecylammonium bromide, N , N -dicoctadecyl
  • Immunological agents may include carriers such as thyroglobulin; albumins such as human serum albumin; toxins, toxoids or any mutant crossreactive material (CRM) of the toxin from tetanus, diphtheria, pertussis, Pseudomonas, E. coli, Staphylococcus, and Streptococcus ; polyamino acids such as poly(lysine:glutamic acid); influenza; Rotavirus VP6, Parvovirus VP1 and VP2; hepatitis B virus core protein; and hepatitis B virus recombinant vaccine.
  • a fragment or epitope of a carrier protein or other immunogenic protein may be used.
  • a T cell epitope of a bacterial toxin, toxoid or CRM may be used.
  • U.S. Patent No 5,785,973 not according to the invention.
  • compositions and vaccines may be administered to mammals in the form of attenuated or inactivated bacteria that may be genetically modified to express the J8 peptide, fragment, variant or derivative and/or the agent that facilitates restoring or enhancing neutrophil activity.
  • attenuated bacteria include Salmonella species, for example Salmonella enterica var. Typhimurium or Salmonella typhi.
  • enteric pathogens such as Shigella species or E. coli may be used in attenuated form.
  • Attenuated Salmonella strains have been constructed by inactivating genes in the aromatic amino acid biosynthetic pathway ( Alderton et al., Avian Diseases 35 435 ), by introducing mutations into two genes in the aromatic amino acid biosynthetic pathway (such as described in U.S. patent 5,770,214 ) or in other genes such as htrA (such as described in U.S. patent 5,980,907 ) or in genes encoding outer membrane proteins, such as ompR (such as described in U.S. patent 5,851,519 ).
  • any safe route of administration may be employed, including oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intra-muscular, intra-dermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular, topical, mucosal and transdermal administration.
  • dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, nasal sprays, suppositories, aerosols, and transdermal patches.
  • dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion.
  • Controlled release may be effected by coating with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose.
  • the controlled release may be effected by using other polymer matrices, liposomes and/or microspheres.
  • compositions may be presented as discrete units such as capsules, sachets, functional foods/feeds or tablets each containing a pre-determined amount of one or more therapeutic agents as described herien, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion.
  • Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more agents as described above with the carrier which constitutes one or more necessary ingredients.
  • the compositions are prepared by uniformly and intimately admixing the agents as described herein with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.
  • compositions may be administered in a manner compatible with the dosage formulation, and in such amount as effective.
  • the dose administered to a patient should be sufficient to effect a beneficial response in a patient over an appropriate period of time.
  • the quantity of agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof, factors that will depend on the judgement of the practitioner.
  • the terms " patient " , " individual “ and “ subject” are used in the context of any mammalian recipient of a treatment or composition disclosed herein. Accordingly, the methods and compositions disclosed herein may have medical and/or veterinary applications.In this regard, the mammal may be a human.
  • mice BALB/c mice (female, 4-6 weeks old) were sourced from Animal Resource centre (ARC, Perth, Western Australia). All protocols were approved by Griffith University's Animal Ethics Committee in accordance with the National Health and Medical Research Council (NHMRC) of Australia guidelines.
  • Bacterial strains and culture media A number of GAS isolates obtained from various sources were utilized in the study. S. pyogenes M1 ( emm 1) , 88/30 ( emm 97), BSA10 ( emm 124) and NS27 ( emm 91), NS1 ( emm 100) and 90/31 ( emm 57 ) were obtained from Menzies School of Health Research, Darwin, Australia.
  • the strain 5448AP ( emm 1) was obtained from Prof Mark Walker's (University of Queensland, Brisbane, Australia) lab. All strains were passaged in mice and made streptomycin (200 ⁇ g/ml) resistant by continually replating them on increasing concentrations of streptomycin.
  • streptomycin 200 ⁇ g/ml
  • streptomycin 200 ⁇ g/ml
  • TAB Todd-Hewitt broth
  • neopeptone 1% neopeptone
  • Peptide synthesis and vaccine formulation Peptide J8 was synthesized and conjugated to DT by Auspep Pty Ltd (Australia) as described elsewhere (Batzloff et al, 2003). The recombinant SpyCEP was expressed and purified by GenScript USA Inc. All peptides were stored lyophilized or in solution at -80 0 C. Mice were vaccinated with J8-DT or recombinant SpyCEP at a dose of 30 ⁇ g/mouse.
  • mice To develop a superficial skin infection model for GAS, inbred female BALB/c, and outbred Swiss mice (4-6 weeks old) were used. Mice were anesthetized with an intraperitoneal (IP) injection (100 ⁇ l/10g mouse) of Ketamine (100 mg/ml stock)/Xylazil-20 (20 mg/ml stock)/water in a ratio 1:1:10. The furs from the back haunch of mice were removed using clippers and shaver. The skin was wiped clean with an ethanol swab and then mechanically scarified with the help of a metal file. Following a skin abrasion, the mice were infected with GAS.
  • IP intraperitoneal
  • mice were immunized subcutaneously at the tail base on day 0 with 30 ⁇ g of J8-DT, 30 ⁇ g of rSpyCEP or 60 ⁇ g formulation containing 30 ⁇ g of J8-DT and 30 ⁇ g of rSpyCEP. All antigen preparations in PBS were formulated in Alhydrogel as an adjuvant. Mice were boosted on day 21 and 28. Control mice received adjuvant alone. Two weeks after the final immunization, mice were challenged with GAS using skin route of infection. Post-infection the mice were monitored closely and any mice showing signs of illness (based on score sheet, approved by GU animal ethics committee) were sacrificed.
  • Sample collection and CFU determination At defined time-points post-infection (day 3, 6 and 9) 5 mice from each group were culled. Blood samples were collected via cardiac puncture and skin tissue was excised from the infected lesions at the back haunch of the infected mice. The skin samples were weighed and homogenized in saline. Appropriate dilutions were then plated in replicates on Streptomycin-blood-agar plates to determine bacterial load in the infected lesion. Blood samples were diluted in PBS and appropriate dilutions were plated in replicates on streptomycin-blood-agar plates to determine bacterial load in the blood.
  • Macrophages were depleted via IP administration of carrageenan (CGN) as previously described (Goldmann et al., 2004).
  • CGN carrageenan
  • the dose and time-course for CGN injection was optimized using flow cytometric analysis of spleen cells following labeling with FITC-conjugated anti-mouse Mac-1 and APC conjugated F4/80 (BD Biosciences, New Jersey, USA).
  • anti-Ly6G mAb clone 1A8 were used as previously described (Eyles, et al., 2008). The depletion of neutrophils was confirmed by flow cytometric analysis of blood, bone-marrow and spleen cells using CD11b-perCp-cy5.5 and Gr-1-APC mAbs.
  • IL-8, MIP-2 and KC degradation were performed and quantified by ELISA using the Quantikine kit (R & D systems, Minneapolis, MN, USA) as described previously (Hidalgo-Grass et al., 2004). Using this method the amounts of undegraded chemokines (IL-8, MIP-2 and KC) post incubation with GAS culture supernatants (S/N) were measured. Briefly, to collect culture S/N, various GAS strains were grown to mid-log phase (OD 600 0.5), re-inoculated into fresh THB and grown overnight at 37 0 C.
  • Neutrophil isolation and transwell migration assays Neutrophils were isolated from mouse bone-marrow using neutrophil isolation kit (Miltenyi Biotech, Germany). Neutrophils (2.5 ⁇ 10 5 in 100 ⁇ l media) were added to the upper chamber of the transwell system (Costar 24-well transwell, Corning NY), which was then placed in the lower chamber containing media alone or intact or degraded chemokines. As a positive control, wells containing known concentration of each recombinant chemokine were also used. Following 2 h of incubation at 37 0 C, the cells were collected from both upper and lower chambers and the number of viable neutrophils transmigrated were determined using trypan blue exclusion.
  • SpyCEP neutralization assays To assess the SpyCEP neutralizing ability of rSpyCEP antisera, hyper-immune serum to rSpyCEP was raised in BALB/C mice. A panel of GAS strains including 90/31, BSA10 and 5448AP were grown to stationary phase. The cell free GAS culture supernatants were co-incubated with recombinant chemokines and either 50% normal or anti-SpyCEP serum for 16 h at 37 0 C. Uncleaved IL-8 was measured using a Quantikine ELISA kit (R & D Systems).
  • SpyCEP Epitope mapping A peptide array encompassing 553 amino acids from the N-terminal region of SpyCEP was synthesized at GenScript (Genscript USA Inc). In total 55 peptides (SEQ ID NOs: 1-55), each 20-mer overlapping by 10 were blotted on to the membrane. Following an ELISA protocol the membrane was probed with antisera from mice immunized 3 times with 30 ⁇ g/dose of rSpyCEP/Alum preparation. Six peptides that were recognized most strongly with SpyCEP antisera were taken for further studies (SEQ ID NOs: 15, 18, 19, 24, 30 and 54).
  • Peptide synthesis and vaccine formulation Six peptides (20-mer each) identified by epitope mapping (as discussed above) were synthesized at GenScript either as a free peptide or with an additional Cysteine residue at the C-terminus. The peptides with C-terminus were then conjugated to DT and were used in mice for in vivo immunogenicity studies. All peptides were stored lyophilized or in solution at -20 0 C. Mice were vaccinated 3 times with rSpyCEP or SpyCEP peptide (S1-S6)-DT conjugates at a dose of 30 ⁇ g/mouse.
  • Serum samples were collected before and after each boost to determine antibody titers to the immunizing peptides as well as to the parent protein (rSpyCEP).
  • rSpyCEP the parent protein
  • ELISA was carried out using 2-fold serial dilutions of antiserum raised against each peptide.
  • SpyCEP peptide recognition by rSpyCEP two-fold serial dilution of rSpyCEP antiserum was used.
  • parent peptide recognition of peptide anti-sera the plates were coated with rSpyCEP and peptide antisera was used to assess its binding/recognition of rSpyCEP.
  • Assay for IL-8 protection in vitro To assess the ability of peptide antisera in inhibiting IL-8 degradation, an in vitro IL-8 protection assay was performed. GAS culture supernatants (S/N) were incubated with a known concentration of recombinant IL-8 and 1:2 dilution of peptide antisera (S1-S6). The amount of undegraded IL-8 in the reaction mixture was quantified by ELISA using the Quantikine kit (R & D systems, Minneapolis, MN, USA) as described previously (Hidalgo-Grass et al., 2004).
  • Peptide inhibition ELISA For the peptide inhibition ELISA assays, the microtiter plates were coated with 100 ⁇ l of peptide S1 to S6 or recSpyCEP at a final concentration of 5 ⁇ g/ml in 75 mM sodium carbonate buffer, pH 9.6, at 4 0 C overnight. Plates were washed with buffer (PBS with 0.05 % Tween 20, pH 7.2) and blocked for 90 min at 37°C with buffer supplemented with 5 % skim milk (blocking buffer). Anti-peptide or recSpyCEP sera were pre-incubated with 5 or 2.5 ⁇ g/ml of each of the self-peptide or recSpyCEP for 30 min at 37 0 C.
  • buffer PBS with 0.05 % Tween 20, pH 7.2
  • Test antisera were thereafter added to the blocked plates and incubated for 90 min at 37°C. The plates were washed four times and HRP-conjugated goat anti-mouse-IgG (Biorad, Australia) was added and incubated for another 90 min at 37°C. After additional washing, the plates were developed as above and OD 450 measured.
  • Flow cytometry assay The binding of the recSpyCEP or SpyCEP epitope antibodies to GAS cell surface was analysed by flow cytometry. The bacteria were grown in THB with 1% neopeptone overnight and washed in PBS.
  • bacteria (1 ⁇ 10 7 colony forming units; cfu) were pre-incubated with 100 ⁇ l of Fc blocker for 15 min at RT to block the non-specific binding sites. This was followed by addition of the peptide antiserum at a dilution of 1 in 20. After 1 h incubation at RT, the bacteria were washed twice in PBS followed by incubation with a FITC-conjugated anti-mouse IgG (diluted 1/50 in PBS with 2 % BSA). Finally, the bacteria were washed and incubated in 1 % formaldehyde (in PBS) for 15 min at RT.
  • the samples were analysed in a CyAn ADP Analyzer (Beckman Coulter, Inc.).
  • the FITC-conjugated anti-mouse IgG was added separately as a negative control for each strain analyzed and in addition, a non-specific mouse IgG was included as a control.
  • mice were fed on streptomycin (200 ⁇ g/ml) water 24 h prior to infection and remained on that throughout the course of study. Mice were monitored daily for infected lesions as well as signs of illness as per the score sheet approved by Griffith University IBC. The wounded site was closely monitored to evaluate the status of infection.
  • Organ collection and CFU determination At various time-points post-infection (day 3, 6 and 9) a defined number of mice from each group were sacrificed. Blood samples were collected via cardiac puncture, spleens were removed and the skin biopsy samples from the infected lesion at the nape of the neck were obtained. The skin and spleen samples were homogenized and appropriate dilutions were then plated in replicates on streptomycin-blood-agar plates. Post-infection the mice were monitored closely and any mice showing signs of illness (based on a score sheet) were sacrificed.
  • FIG 1 the protective efficacy of J8-DT/Alum vaccination is shown by comparing different GAS strains M1 (a throat isolate) and skin isolates 88/30 and BSA10. J8-DT/Alum vaccination was less effective for both of the skin isolates compared to M1. Associated with the effectiveness of J8 immunization is the presence of neutrophils (PMN) in the skin of GAS infected mice, as shown in Figure 2 .
  • PMN neutrophils
  • FIG. 4 The data in Figures 1-3 suggested that neutrophils may play a role in determining the efficacy of J8 immunization against GAS infection.
  • Figure 4 provides an overview of the bacterial genes that are expressed upregulated in GAS isolates such as 5448AP (M1T1 isolate) that have a CovR/S mutation.
  • the efficacy of J8-DT/Alum protection against GAS isolate 5448AP was relatively poor.
  • a candidate bacterial gene that is highly expressed in GAS isolate 5448AP was SpyCEP, a 70 kD serine protease that cleaves and inactivates the neutrophil chemoattractant interleukin 8 (see FIG. 4 ).
  • FIG 6 shows the results of experiments where 5448AP displayed particularly strong IL-8 degradation.
  • the 5448AP isolate also displayed strong degrading activity toward the murine functional homologues of IL-8: CXCL1/MIP-2 ( Figure 7 ) and CXCL1/KC ( Figure 8 ). Functionally, this degradation correlated with an inhibition neutrophil chemotaxis as shown in Figure 9 .
  • Hyperimmune sera were generated in 4-6week old female Balb/c mice following a 28 day immunisation protocol. Mice were immmunised subcutaneously with 30 ⁇ g of DT conjugated peptide (J8, p145, J8iV1, J8iV2, J8iV3 or J8iV4) in PBS and Alum. Immunisations occurred on days 0, 21, 28. Submandibular bleeds to collect blood/sera were performed at days 35, 42 and 49.
  • FIG. 18 shows that the titer of p145-specific antibodies induced by J8-DT vaccination is very low (mean about 2000, with some mice below 200). By contrast, the titer of J8-specific antibodies is about 600,000 following J8-DT vaccination.
  • the high titer to J8 must mean that most antibodies are recognizing the non-streptococal flanking sequences at the amino-terminal and carboxyl-terminal segments of J8, although this has not been formally proven.
  • FIG. 19 shows that J8iV1, J8iV2, J8iV3 and J8iV4 all induce high titer antibodies to themselves (approx. 100,000). Importantly, all induce high titers to p145 (>10,000) with J8iV3-DT inducing titers of approximately 30,000 ( FIG. 20 , right panel). It is probable that the titers of the new peptides to themselves are higher than the titers to p145 because p145 contains additional amino acids not found in the new peptides and some of these may mask the epitopes recognized by the antisera to the new variants. However, the high titers of the variant J8 peptides to themselves (approx.
  • the immunodominance of S2 was further assessed using a peptide inhibition assay.
  • Antisera to recSpyCEP and the individual epitopes were pre-incubated with each of the immunizing antigens after which their binding to the antigen was analysed.
  • the binding of epitope antisera to immobilised self-peptides was greatly reduced following pre-incubation with self-peptide (40-80% inhibition) ( Figure 21 A) .
  • S2 SEQ ID NO: 18
  • S2 SEQ ID NO: 18
  • the inventors then tested the binding efficiency of SpyCEP and epitope antisera to various GAS strains.
  • SpyCEP is expressed on the surface of GAS and also shed.
  • To gauge recognition of native antigen the binding of the different epitope specific antibodies to various GAS strains were compared using a FACS assay.
  • GAS strain 5448 was used alongside with its animal passaged derivative 5448AP (a CovR/S mutant strain known to express high levels of SpyCEP).
  • wild-type BSA10 was used in parallel with its animal passaged derivative pBSA10 (also a CovR/S mutant).
  • Vaccination with J8-DT+S2-DT resulted in significantly reduced bacterial bio-burden in skin as well as in the blood of 5448AP-infected mice ( Figure 23 A and B ).
  • the level of protection offered by J8-DT+S2-DT vaccination was also found to be comparable to that of J8-DT+SpyCEP vaccination.
  • the challenge experiments were repeated with another CovR/S mutant strain NS88.2 ( Figure 24 A and B ).
  • the inventors found that vaccination with J8-DT+SpyCEP or J8-DT+S2-DT results in significantly enhanced protective efficacy compared to J8-DT alone.
  • Vaccination with either J8-DT or SpyCEP alone did not offer any protection in comparison to the PBS controls.
  • SpyCEP is an IL-8 degrading protease highly expressed by virulent GAS bacteria such as those having the CovR/S mutation.
  • Immunization with J8 peptide and rSpyCEP in combination had a synergistic effect against GAS infection by the CovR/S GAS mutant 5448AP compared to J8 peptide or SpyCEP alone. It is also evident that anti-SpyCEP antibodies may effectively neutralize the IL-8 degrading activity of SpyCEP, thereby providing a therapeutic intervention for existing GAS infections.
  • the inventors have identified the dominant epitope on SpyCEP (SEQ ID NO: 18) that can induce functional antibodies.
  • the inventors are currently planning active and passive vaccine studies with GAS challenge.
  • the advantage of this epitope is that it enables us to avoid using whole recombinant SpyCEP protein in the vaccine, thus improving the safety profile.
  • the inventors believe that the optimal vaccine will be a mixture of J8 and this epitope.
  • vaccination with J8-DT+SpyCEP or J8-DT+S2-DT results in significantly enhanced protective efficacy compared to J8-DT alone.
  • variant J8 peptides have been developed which induce high titers to themselves and the streptococcal peptide p145, presumably because they are exclusively derived from streptococcal p145 amino acid sequence.

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Claims (15)

  1. Composition comprenant :
    un fragment de protéine M immunogène :
    (i) constitué du peptide p145 de SEQ ID NO : 56,
    (ii) consistant en un variant de (i) consistant en une séquence d'acides aminés identique à au moins 90 % à SEQ ID NO : 56,
    (iii) comprenant le peptide J8 de SEQ ID NO : 58,
    (iv) comprenant un variant de (iii) consistant en une séquence d'acides aminés identique à au moins 90 % à SEQ ID NO : 58,
    (v) comprenant le peptide variant J8 de SEQ ID NO : 59, SEQ ID NO : 60, SEQ ID NO : 61, ou SEQ ID NO : 62,
    (vi) comprenant un variant de (v) consistant en une séquence d'acides aminés identique à au moins 90 % à celle-ci ;
    (vii) comprenant un dérivé de l'un quelconque des éléments (i) à (vi) consistant en un ou plusieurs résidus lysine à son extrémité N- et/ou C-terminale ; ou
    (viii) comprenant un dérivé conjugué de l'un quelconque des éléments (i) à (vii), consistant en l'un quelconque des éléments (i) à (vii) conjugué à une protéine porteuse, à un fragment ou à un épitope de celle-ci, dans lequel ladite protéine porteuse ne comprend pas de séquence de la protéine M, et
    un agent qui facilite la restauration ou l'augmentation de l'activité des neutrophiles, l'agent comprenant :
    (a) le peptide SpyCEP de SEQ ID NO : 18,
    (b) un variant du peptide SpyCEP consistant en une séquence d'acides aminés au moins 90 % identique à SEQ ID NO : 18,
    (c) le peptide SpyCEP de (a) ou le variant du peptide SpyCEP de (b) consistant en outre en un ou plusieurs résidus lysine à son extrémité N- et/ou C-terminale ;
    (d) le peptide SpyCEP ou le variant du peptide SpyCEP de l'un quelconque de (a) à (c) conjugué à une protéine porteuse, à un fragment ou à un épitope de celle-ci ; ou
    (e) un anticorps ou un fragment d'anticorps qui lie le peptide SpyCEP ou le variant du peptide SpyCEP de l'un quelconque de (a) à (d),
    pour utilisation dans un procédé visant à déclencher une réponse immunitaire aux bactéries streptococciques du groupe A chez un mammifère.
  2. Composition comprenant :
    un fragment de protéine M immunogène :
    (i) constitué du peptide p145 de SEQ ID NO : 56,
    (ii) consistant en un variant de (i) consistant en une séquence d'acides aminés identique à au moins 90 % à SEQ ID NO : 56,
    (iii) comprenant le peptide J8 de SEQ ID NO : 58,
    (iv) comprenant un variant de (iii) consistant en une séquence d'acides aminés identique à au moins 90 % à SEQ ID NO : 58,
    (v) comprenant le peptide variant J8 de SEQ ID NO : 59, SEQ ID NO : 60, SEQ ID NO : 61, ou SEQ ID NO : 62,
    (vi) comprenant un variant de (v) consistant en une séquence d'acides aminés identique à au moins 90 % à celle-ci ;
    (vii) comprenant un dérivé de l'un quelconque des éléments (i) à (vi) consistant en un ou plusieurs résidus lysine à son extrémité N- et/ou C-terminale ; ou
    (viii) comprenant un dérivé conjugué de l'un quelconque des éléments (i) à (vii), consistant en l'un quelconque des éléments (i) à (vii) conjugué à une protéine porteuse, à un fragment ou à un épitope de celle-ci, dans lequel ladite protéine porteuse ne comprend pas de séquence de la protéine M, et
    un agent qui facilite la restauration ou l'augmentation de l'activité des neutrophiles, l'agent comprenant :
    (a) le peptide SpyCEP de SEQ ID NO : 18,
    (b) un variant du peptide SpyCEP consistant en une séquence d'acides aminés au moins 90 % identique à SEQ ID NO : 18,
    (c) le peptide SpyCEP de (a) ou le variant du peptide SpyCEP de (b) consistant en outre en un ou plusieurs résidus lysine à son extrémité N- et/ou C-terminale ;
    (d) le peptide SpyCEP ou le variant du peptide SpyCEP de l'un quelconque de (a) à (c) conjugué à une protéine porteuse, à un fragment ou à un épitope de celle-ci ; ou
    (e) un anticorps ou un fragment d'anticorps qui lie le peptide SpyCEP ou le variant du peptide SpyCEP de l'un quelconque de (a) à (d),
    pour utilisation dans un procédé d'immunisation d'un mammifère contre les bactéries streptococciques du groupe A.
  3. Composition comprenant :
    un fragment de protéine M immunogène :
    (i) constitué du peptide p145 de SEQ ID NO : 56,
    (ii) consistant en un variant de (i) consistant en une séquence d'acides aminés identique à au moins 90 % à SEQ ID NO : 56,
    (iii) comprenant le peptide J8 de SEQ ID NO : 58,
    (iv) comprenant un variant de (iii) consistant en une séquence d'acides aminés identique à au moins 90 % à SEQ ID NO : 58,
    (v) comprenant le peptide variant J8 de SEQ ID NO : 59, SEQ ID NO : 60, SEQ ID NO : 61, ou SEQ ID NO : 62,
    (vi) comprenant un variant de (v) consistant en une séquence d'acides aminés identique à au moins 90 % à celle-ci ;
    (vii) comprenant un dérivé de l'un quelconque des éléments (i) à (vi) consistant en un ou plusieurs résidus lysine à son extrémité N- et/ou C-terminale, ou
    (viii) comprenant un dérivé conjugué de l'un quelconque des éléments (i) à (vii), consistant en l'un quelconque des éléments (i) à (vii) conjugué à une protéine porteuse, à un fragment ou à un épitope de celle-ci, dans lequel ladite protéine porteuse ne comprend pas de séquence de la protéine M,
    ou
    un anticorps ou un fragment d'anticorps qui se lie au fragment de protéine M,
    et
    un agent qui facilite la restauration ou l'augmentation de l'activité des neutrophiles, l'agent comprenant :
    (a) le peptide SpyCEP de SEQ ID NO : 18,
    (b) un variant du peptide SpyCEP consistant en une séquence d'acides aminés au moins 90 % identique à SEQ ID NO : 18,
    (c) le peptide SpyCEP de (a) ou le variant du peptide SpyCEP de (b) consistant en outre en un ou plusieurs résidus lysine à son extrémité N- et/ou C-terminale ;
    (d) le peptide SpyCEP ou le variant du peptide SpyCEP de l'un quelconque de (a) à (c) conjugué à une protéine porteuse, à un fragment ou à un épitope de celle-ci ; ou
    (e) un anticorps ou un fragment d'anticorps qui lie le peptide SpyCEP ou le variant du peptide SpyCEP de l'un quelconque de (a) à (d),
    pour utilisation dans un procédé de traitement ou de prévention d'une infection bactérienne à streptocoques du groupe A chez un mammifère.
  4. Composition comprenant un ou plusieurs acides nucléiques isolés codant :
    un fragment de protéine M immunogène :
    (i) constitué du peptide p145 de SEQ ID NO : 56,
    (ii) consistant en un variant de (i) consistant en une séquence d'acides aminés identique à au moins 90 % à SEQ ID NO : 56,
    (iii) comprenant le peptide J8 de SEQ ID NO : 58,
    (iv) comprenant un variant de (iii) consistant en une séquence d'acides aminés identique à au moins 90 % à SEQ ID NO : 58,
    (v) comprenant le peptide variant J8 de SEQ ID NO : 59, SEQ ID NO : 60, SEQ ID NO : 61, ou SEQ ID NO : 62,
    (vi) comprenant un variant de (v) consistant en une séquence d'acides aminés identique à au moins 90 % à celle-ci ;
    (vii) comprenant un dérivé de l'un quelconque des éléments (i) à (vi) consistant en un ou plusieurs résidus lysine à son extrémité N- et/ou C-terminale ; ou
    (viii) comprenant un dérivé conjugué de l'un quelconque des éléments (i) à (vii), consistant en l'un quelconque des éléments (i) à (vii) conjugué à une protéine porteuse, à un fragment ou à un épitope de celle-ci, dans lequel ladite protéine porteuse ne comprend pas de séquence de la protéine M,
    et
    un agent qui facilite la restauration ou l'augmentation de l'activité des neutrophiles, l'agent comprenant :
    (a) le peptide SpyCEP de SEQ ID NO : 18,
    (b) un variant du peptide SpyCEP consistant en une séquence d'acides aminés au moins 90 % identique à SEQ ID NO : 18,
    (c) le peptide SpyCEP de (a) ou le variant du peptide SpyCEP de (b) consistant en outre en un ou plusieurs résidus lysine à son extrémité N- et/ou C-terminale ; ou
    (d) le peptide SpyCEP ou le variant quelconque du peptide SpyCEP de l'un de (a) à (c) conjugué à une protéine porteuse, à un fragment ou à un épitope de celle-ci,
    pour utilisation dans un procédé visant à déclencher une réponse immunitaire aux bactéries streptococciques du groupe A chez un mammifère, pour utilisation dans un procédé d'immunisation d'un mammifère contre les bactéries streptococciques du groupe A, ou pour utilisation dans un procédé de traitement ou de prévention d'une infection bactérienne à streptocoques du groupe A chez un mammifère.
  5. Composition appropriée pour être administrée à un mammifère, ladite composition comprenant :
    un fragment de protéine M immunogène :
    (i) constitué du peptide p145 de SEQ ID NO : 56,
    (ii) consistant en un variant de (i) consistant en une séquence d'acides aminés identique à au moins 90 % à SEQ ID NO : 56,
    (iii) comprenant le peptide J8 de SEQ ID NO : 58,
    (iv) comprenant un variant de (iii) consistant en une séquence d'acides aminés identique à au moins 90 % à SEQ ID NO : 58,
    (v) comprenant le peptide variant J8 de SEQ ID NO : 59, SEQ ID NO : 60, SEQ ID NO : 61, ou SEQ ID NO : 62,
    (vi) comprenant un variant de (v) consistant en une séquence d'acides aminés identique à au moins 90 % à celle-ci ;
    (vii) comprenant un dérivé de l'un quelconque des éléments (i) à (vi) consistant en un ou plusieurs résidus lysine à son extrémité N- et/ou C-terminale ; ou
    (viii) comprenant un dérivé conjugué de l'un quelconque des éléments (i) à (vii), consistant en l'un quelconque des éléments (i) à (vii) conjugué à une protéine porteuse, à un fragment ou à un épitope de celle-ci, dans lequel ladite protéine porteuse ne comprend pas de séquence de la protéine M,
    ou
    un anticorps ou un fragment d'anticorps qui se lie au fragment de protéine M,
    et
    un agent qui facilite la restauration ou l'augmentation de l'activité des neutrophiles, l'agent comprenant :
    (a) le peptide SpyCEP de SEQ ID NO : 18,
    (b) un variant du peptide SpyCEP consistant en une séquence d'acides aminés au moins 90 % identique à SEQ ID NO : 18,
    (c) le peptide SpyCEP de (a) ou le variant du peptide SpyCEP de (b) consistant en outre en un ou plusieurs résidus lysine à son extrémité N- et/ou C-terminale ;
    (d) le peptide SpyCEP ou le variant du peptide SpyCEP de l'un quelconque de (a) à (c) conjugué à une protéine porteuse, à un fragment ou à un épitope de celle-ci ; ou
    (e) un anticorps ou un fragment d'anticorps qui lie le peptide SpyCEP ou le variant du peptide SpyCEP de l'un quelconque de (a) à (d).
  6. Composition appropriée pour être administrée à un mammifère, ladite composition comprenant un ou plusieurs acides nucléiques isolés codant :
    un fragment de protéine M immunogène :
    (i) constitué du peptide p145 de SEQ ID NO : 56,
    (ii) consistant en un variant de (i) consistant en une séquence d'acides aminés identique à au moins 90 % à SEQ ID NO : 56,
    (iii) comprenant le peptide J8 de SEQ ID NO : 58,
    (iv) comprenant un variant de (iii) consistant en une séquence d'acides aminés identique à au moins 90 % à SEQ ID NO : 58,
    (v) comprenant le peptide variant J8 de SEQ ID NO : 59, SEQ ID NO : 60, SEQ ID NO : 61, ou SEQ ID NO : 62,
    (vi) comprenant un variant de (v) consistant en une séquence d'acides aminés identique à au moins 90 % à celle-ci ;
    (vii) comprenant un dérivé de l'un quelconque des éléments (i) à (vi) consistant en un ou plusieurs résidus lysine à son extrémité N- et/ou C-terminale ; ou
    (viii) comprenant un dérivé conjugué de l'un quelconque des éléments (i) à (vii), consistant en l'un quelconque des éléments (i) à (vii) conjugué à une protéine porteuse, à un fragment ou à un épitope de celle-ci, dans lequel ladite protéine porteuse ne comprend pas de séquence de la protéine M,
    et
    un agent qui facilite la restauration ou l'augmentation de l'activité des neutrophiles, l'agent comprenant :
    (a) le peptide SpyCEP de SEQ ID NO : 18,
    (b) un variant du peptide SpyCEP consistant en une séquence d'acides aminés au moins 90 % identique à SEQ ID NO : 18,
    (c) le peptide SpyCEP de (a) ou le variant du peptide SpyCEP de (b) consistant en outre en un ou plusieurs résidus lysine à son extrémité N- et/ou C-terminale ; ou
    (d) le peptide SpyCEP ou le variant du peptide SpyCEP de l'un quelconque de (a) à (c) conjugué à une protéine porteuse, à un fragment ou à un épitope de celle-ci.
  7. Composition pour utilisation selon l'une quelconque des revendications 1 à 3, dans laquelle l'agent qui facilite la restauration ou l'amélioration de l'activité des neutrophiles comprend le peptide SpyCEP ou le variant du peptide SpyCEP.
  8. Composition pour utilisation selon l'une quelconque des revendications 1 à 3, dans laquelle l'agent qui facilite la restauration ou l'amélioration de l'activité des neutrophiles comprend ledit anticorps ou fragment d'anticorps, qui se lie au peptide SpyCEP ou au variant du peptide SpyCEP selon l'un quelconque de (a) à (d).
  9. Composition pour utilisation selon l'une quelconque des revendications 1 à 4, 7 et 8, dans laquelle le mammifère est un humain.
  10. Composition pour utilisation selon l'une quelconque des revendications 1 à 4 et 7 à 9, dans laquelle ladite protéine porteuse comprend la thyroglobuline, l'albumine, une toxine, une anatoxine ou toute substance mutante à réaction croisée (CRM), de préférence de la toxine du tétanos, de la diphtérie, de la coqueluche, de Pseudomonas, d'E. coli, de Staphylococcus ou de Streptococcus, ou un fragment ou épitope de celle-ci, ou ladite protéine porteuse comprend une séquence d'acides aminés non trouvée dans les streptocoques du groupe A A , ou ladite protéine porteuse est l'anatoxine diphtérique.
  11. Composition pour utilisation selon l'une quelconque des revendications 1 à 4 et 7 à 10, dans laquelle le peptide SpyCEP ou le variant de (c) comprend un ou plusieurs résidus lysine à son extrémité N-terminale.
  12. Composition selon la revendication 5, dans laquelle l'agent qui facilite la restauration ou l'amélioration de l'activité des neutrophiles comprend le peptide SpyCEP ou son variant de peptide SpyCEP ; ou dans laquelle l'agent qui facilite la restauration ou l'amélioration de l'activité des neutrophiles comprend ledit anticorps ou fragment d'anticorps, qui se lie au peptide SpyCEP ou au variant de peptide SpyCEP de l'un quelconque de (a) à (d).
  13. Composition selon l'une quelconque des revendications 5, 6 et 12, dans laquelle ladite protéine porteuse comprend la thyroglobuline, l'albumine, une toxine, une anatoxine ou toute substance mutante à réaction croisée (CRM), de préférence de la toxine du tétanos, de la diphtérie, de la coqueluche, de Pseudomonas, d'E. coli, de Staphylococcus ou de Streptococcus, ou un fragment ou épitope de celle-ci, ou ladite protéine porteuse comprend une séquence d'acides aminés non trouvée dans les streptocoques du groupe A A , ou ladite protéine porteuse est l'anatoxine diphtérique.
  14. Composition pour utilisation selon l'une quelconque des revendications 5, 6, 12 et 13, dans laquelle le peptide SpyCEP ou le variant de peptide SpyCEP de (c) comprend un ou plusieurs résidus lysine à son extrémité N-terminale.
  15. Composition selon l'une quelconque des revendications 5, 6 et 12 à 14, dans laquelle le mammifère est un humain.
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IL248266A0 (en) 2016-11-30
EP3131575A1 (fr) 2017-02-22
JP7072921B2 (ja) 2022-05-23
JP2021042247A (ja) 2021-03-18
IL248266B (en) 2021-09-30
ZA201607355B (en) 2019-03-27
CN106794236A (zh) 2017-05-31
JP2017513849A (ja) 2017-06-01
CA2945052C (fr) 2023-05-02
WO2015157820A1 (fr) 2015-10-22
CN106794236B (zh) 2021-09-03
NZ725212A (en) 2023-04-28
KR20160144455A (ko) 2016-12-16
MY191217A (en) 2022-06-09
AU2015246654A1 (en) 2016-11-03
BR112016023915A2 (pt) 2017-10-17
US10513544B2 (en) 2019-12-24
DK3131575T3 (da) 2024-05-21
MX2016013277A (es) 2017-01-18
KR20230039751A (ko) 2023-03-21
US20170037087A1 (en) 2017-02-09
CA2945052A1 (fr) 2015-10-22
US20190256562A1 (en) 2019-08-22
EP3131575A4 (fr) 2017-11-01

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